Soluble protamine insulin



Pi nemsisnn; it 2,190,137

SOLUBLE PR TA INE INSULIN 3 .t Melville Sahyun, Detroit,,Mioh., assignor to' FrederickStearns 85 Company, Detroit, Mich., a

corporation oLMichigan' o No Drawings Application July 10, 1937, 1 lSerial'No. 152,995 N m6 Claims; (01.167-75) p .1 A This invention relates to water soluble protapost-prandial rise in thejblood sugar takesfplace mine'insulin and the process of producing the afterfits" administration necessitating a slmul same, l V V taneous injectionof regular insulin to compen- By this invention protamine insulin can-be profe for thi defi i y. duced in a very rapid and convenient manner 1 (e) In view of the factthat such aprecipi= 5 without requiring expensive apparatus and withtated material has a hydrogen ion concentration Outas much difliculty as is experienced in prosuitable for bacterial growth, the utmost preducing insoluble protamine insulin that is now auti n is necessary t s e mple Sterility. on the market. The protamine insulin of this infor unless th fi P p t is perfectly clear, 10' 'vention possesses the prolonged hypoglycemi efit; is extremely diflicult to know, by mere inspec- 10 feet, that has been, observed t msoluble s tion of the vial, whether or not the material has tamine insulin and at thesame time it'has all becQme contaminated y a. the advantages due to its being soluble in water. In the preparation i the precipitated pr a- It is w n known t t t mi g t of mine-insulin complex of the prior art, 0.75 tol.25 insulin causes a very rapid lowering or blood mi a s o p ne we eu d pe 100 units sugar with subsequent rapid return to the initial of insulin. diSSOll/ed. in a and about 1 0f level., From clinical and animal experimentaa 2 P cent Solution q 'd hydrogen Ph tionllt has been found thatthe effect of insulin photo was added to raisethe oge ion conon blood sugar does not usually last more than fi l' 0f insulin a d nr t 2o 6 or 'lhours, so that many diabetic patients need l f fi ut P t was found t t Such 20 V to be given as many asfour injections of insulin I addltlon O Protamine o insulin. n the s b daily. investigators have therefore been work quell? injection as a clear Solution d P omg diligently w d t preparation of a mduce a more prolonged action on the blood sugar mm t t, would exert prolgnged hypogwcemic than that of regular insulin but that the product effect in the system, thus reducing the number had to beprecimtated in order for i o pr duce 5 of necessary daily injections. Emulsions of ina prolonged hypoglycemic e c This precipi sulin with oil have been tried and have been found ated prod 15 I10W on h e to produce a somewhat more prolonged hypo- By the P t invention I use a method that glycemic effect than regular insulin. However, enables me to P e a C Solutionflf 1 18111 since such preparations were nothomogeneous Protamine complex P ep that is 9411116317 they were not satisfactory to the clinicians and as rapid initially in its c n s regular insulin the diabetic patients. and yet as prolonged in its effect as the .preci- It has also been known for some time that so- Pita/Bed -p a ne; caged t m insulin could be produced by g In myinvestigations of the efiect of protamine' precipitating insulin from a water solution by the on insulin with reference to the prolongation of 35 addition of protamineand that the precipitated the action of the latter, I have found that by insulin-protainine complex, when administered using 2.5 to.'5.0 rnilligramsof protamine to 100 to patients, acted more slowly on the blood sugar units of water'solution of insulin, and by heating of t sy m d maintained a w lood su the insulin and protami'ne and allowingjthe mix- 40 level a longer Period tune than the 8 ture to stand for several days, a complex or l (1085- of i @aflvantages of. preparation is formed that has an initial rapid suchprec pitated protaminee nsulln preparations hypoglmemic effect and yet the duration of or complexes are: I hypoglycemia is greatlyprolonged. Slim.

(a) The are not 'sable over lon riod of V 7, time and {heremre exmmtma'n i g be preparation has been carefully studiedon animals e i and human beings and has been found to possess placed on each package of them.

(1,), such preparations do not remain hom0ge the following distinct advantages over other pronegus upon standing atroom temperature, and tamine-insulin preparations now on the market: quite often a sticky material is formed on the p (a) This new preparation f a lear insulinside f the containers, 7 V protamine solution is permanently stable and 50 (c) It has ben observed that nodules are often therefore does. not require an expiration date to found at the site of injection 01' such protaniinebe placed on the packages thereof. insulin preparations. (b) Its initial action is rapid so that this clear (d) The action of the precipitated protamineinsulin-protamine begins to take eflect at once insulin complex is slow, so that a considerable upon administration, thus eliminating the neces- 55 careful adjustment of the hydrogen ion concen-- tration. I

The danger of bacterial contamination is considerably reduced. My preparation issterilized by'one filtration, whereas withthe prior product, that is, a precipitated or cloudy insulinprotamine, one must'sterilize insulin or'insulin and protamine and introduce them into a sterile vial and then into this vial a sterile solution of pH 3.0 to 3.5.

phosphate buffer must be added for. the adjustment of the hydrogen ion concentration.

(I) The subcutaneous administration of this 'clear solution of insulin-protamine' does not 'cause the formation of nodules at the site of injection.

The following are givenas specific examples to illustrate the invention, but it is to be understood that the invention is not restricted to the specific details or the exact temperatures, ma-' terials, or proportions thereof that are mentioned:

Example I To a known concentration of insulin"5.0 milligrams of a highly purified protamine fol-every 100units of insulin are added. The hydrogen ion concentration need not be carefully adjusted, other than to maintain an acidity slightly below that of the precipitation of insulin, for example, The mixture is then carefully warmed in a water bath'below the temperature at which the insulin protein would bedenatured. I have found that temperatures of about 55-55" C. are suitable for this and that about 2 or 3 hours are need at such temperatures. The volume is adjusted by adding an isotonic medium such as water and glycerine, for example, to produce the desired concentration of units of insulin per 1 cc.

The material is next rendered sterile by any or the usual methods.

a solution of crystalline insulin, approximately 5 milligrams of pure protamine to every 190 ofinsulin are added, followedby a small quantity of an acid phosphate, such as sodium or potassium di-hydrogenphosphate, and about 5.2 mini gram of zinc. Thepurposeof the latter is to 1 preventdeterioration of the potency of the final product. The mixture is then heated to about 50 to 55 0.. for 2 or 3 hours, then kept in an incubator at about 50 C. for approximately as to 48 hours, and isthenbrought to the desired 7 concentration and 'sterilized.

I claim:

l. The process of producing a protamine-insulin that is entirely water soluble which coo-1 prises combining protamine and insulin the proportionsof at least 2.5 mg. of protamine to 100 units of insulin in an aqueous medium whose pH is below 3.5 at a temperature below about '2. The process of producing a protaminedm sulin that entirely water soluble which'com prises combining protarnine and insulin in the proportions of at least 2.5 mg. of protamine to its units of insulin in an aqueous medium whose pH is below 3.5, and heating to about 50 to 55 C.

3. The process of producing a protarnine in sulin that is entirely water soluble which :o.-"n prises combining protamine and insulin in the proportions of at least 2.5 mg. of protarnine to 100 units of insulin in an aqueous medium whose pH is below 3.5, and heating to about 50 to 55 C. for about 2'or 3 hours.

4. Theprooess of producing a protamine-insulin that is entirely water soluble which comprises combining protamine and insulin in the proportions of at least 2.5 mg. of protamine to V 100 units of insulin in an aqueous medium whose pH is below 3.5 in the presence of about 0.2 mg. of zinc per 100 units of insulin at a temperature below about 55 C.

5. A clear stable solution containing insulin to which has been added from 2.5 mg. to 5 mg. oi protamine for each 100 units of the insulin, said solution having an acidity of about pH 3.0.

6. A clear stable solution containing insulin to which has been added zinc and from 2.5 mg. to 5 mg. of protamine for each 100 units of the insulin, said solution having an acidity of about pH 3.0.

MCELVILLE SAHYUN. 

